Newly emerged, rapid acting antidepressant ketamine is able to achieve therapeutic improvement, in part, through stimulating activity of kinase-dependent signaling pathways. However, current investigations are limited to a single or small number of kinases and are unable to detect novel kinases. The goal of the proposed project is to uncover mechanisms underlying ketamine's antidepressant actions through a unique unbiased high-throughput screening. We propose to use a chemical proteomics approach that couples multiplexed inhibitor beads (MIBs) technology with quantitative mass spectrometry (MS) to capture kinases and their substrates that are activated by ketamine and its active metabolite HNK. This approach will open up a new realm of opportunities to link ketamine treatment with its underlying molecular mechanisms at the proteomics level. Kinases and pathways identified through the profiling approach will be subject for verification and in-depth examination. Preliminary application of this MIBs/MS approach replicated well-known molecular signature of ketamine action identified previously, including mTOR and ERK pathways. Furthermore, preliminary results suggest unchecked kinase activity and unappreciated pathways that mediate initial neural responses to ketamine as well as synaptogenesis induced by ketamine as a means of functional restoration. We expect our effort in identifying novel mechanisms underlying ketamine antidepressant actions to provide unmet therapeutic opportunities in treating depression and possibly other mood disorders.